Selective activation of M4 muscarinic acetylcholine receptors reverses MK-801-induced behavioral impairments and enhances associative learning in rodents

Abstract

Positive allosteric modulators (PAMs) of the M4 muscarinic acetylcholine receptor (mAChR) represent a novel approach for the treatment of psychotic symptoms associated with schizophrenia and other neuropsychiatric disorders. We recently reported that the selective M4 PAM VU0152100 produced an antipsychotic drug-like profile in rodents after amphetamine challenge. Previous studies suggest that enhanced cholinergic activity may also improve cognitive function and reverse deficits observed with reduced signaling through the N-methyl-d-aspartate subtype of the glutamate receptor (NMDAR) in the central nervous system. Prior to this study, the M1 mAChR subtype was viewed as the primary candidate for these actions relative to the other mAChR subtypes. Here we describe the discovery of a novel M4 PAM, VU0467154, with enhanced in vitro potency and improved pharmacokinetic properties relative to other M4 PAMs, enabling a more extensive characterization of M4 actions in rodent models. We used VU0467154 to test the hypothesis that selective potentiation of M4 receptor signaling could ameliorate the behavioral, cognitive, and neurochemical impairments induced by the noncompetitive NMDAR antagonist MK-801. VU0467154 produced a robust dose-dependent reversal of MK-801-induced hyperlocomotion and deficits in preclinical models of associative learning and memory functions, including the touchscreen pairwise visual discrimination task in wild-type mice, but failed to reverse these stimulant-induced deficits in M4 KO mice. VU0467154 also enhanced the acquisition of both contextual and cue-mediated fear conditioning when administered alone in wild-type mice. These novel findings suggest that M4 PAMs may provide a strategy for addressing the more complex affective and cognitive disruptions associated with schizophrenia and other neuropsychiatric disorders.

Keywords: Allosteric modulator; M4 muscarinic; MK-801; VU0467154; antipsychotic; cognitive enhancement.

Figure 1

Synthesis and structure of VU0467154. For comparison, the structure of the M₄ PAM VU0152100 is shown as an inset. Reagents and conditions for each step are as follows: (a) methyl 2-mercaptoacetate, sodium hydroxide, methanol, microwave at 150 °C for 30 min; (b) BH₃·THF (borane·tetrahydrofuran), tetrahydrofuran, reflux, ca. 18 h; (c) A + B, HATU, N,N-diisopropyl-ethylamine at room temperature.

Figure 2

VU0467154 potentiates acetylcholine (ACh) responses in M₄-expressing cell lines. (A) VU0467154 is more potent at rat M₄ than VU0152100 and LY2033298. (B) Robust potentiation of an ACh EC₂₀ is seen across species at rat, human, and cynomolgus monkey (cyno) M₄. VU0467154 does not potentiate the ACh response at rat (C) and human (D) M₁, M₂, M₃, or M₅ in calcium mobilization assays. Data were normalized as a percentage of the maximum response to 10 μM ACh and are shown as means ± SEM of at least three independent experiments.

Figure 3

Increasing fixed concentrations of VU0467154 (A) and VU0152100 (C) increase the acetylcholine (ACh) affinity at rat M₄ as measured by ACh-induced displacement of [³H]N-methylscopolamine ([³H]NMS) binding. VU0467154 (B) and VU0152100 (D) cause a progressive leftward shift of an ACh concentration–response curve at rat M₄. Data are shown as means ± SEM of at least three independent experiments.

Figure 4

Time course of plasma and brain levels of VU0467154 following systemic administration to rats and mice. Panels A–D show plasma concentrations after IV (1 mg/kg) and PO (3 and 10 mg/kg) (A) or IP (10 mg/kg) (C) dosing of VU0467154 in rats or following PO (10 and 30 mg/kg) (B) and IP (10 and 30 mg/kg) (D) dosing of the compound in mice. Panel E shows the time course of plasma and brain levels of the compound after IP administration of VU0467154 (3 mg/kg) in mice. Data are means ± SEM (N ≥ 3) or means and individual data points (N = 2).

Figure 5

VU0467154 reverses amphetamine-induced hyperlocomotion in rats. (A) Amphetamine (AMPH) dose-dependently increases open field locomotor activity. Systemic (IP [B] or PO [C]) administration of VU0467154 dose-dependently reverses amphetamine-induced hyperlocomotion. The time course of locomotor activity is shown on the left and total locomotor activity during the 60 min period following amphetamine administration on the right. Data are means ± SEM of 6–8 animals per group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs vehicle (A) or vs vehicle + amphetamine (B and C) (ANOVA followed by Dunnett’s test).

Figure 6

VU0467154 reverses amphetamine-induced hyperlocomotion in wild-type, but not M₄ KO mice. (A) Amphetamine (AMPH) dose-dependently increases open field locomotor activity in wild-type (WT) and M₄ KO mice. (B) In wild-type mice, administration of VU0467154 dose-dependently reverses amphetamine-induced hyperlocomotion. (C) VU0467154 does not reverse amphetamine-induced hyperlocomotion in M₄ KO mice. The time course of locomotor activity is shown on the left and total locomotor activity during the 120 min period following amphetamine administration on the right. Data are means ± SEM of 9–13 wild-type mice and 10–11 M₄ KO mice per group. *** p < 0.001 vs wild-type vehicle + vehicle (A); ^###p < 0.001 vs M₄ KO vehicle + vehicle (A); ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 vs wild-type vehicle + amphetamine (B) (ANOVA followed by Bonferroni’s test).

Figure 7

VU0467154 reverses MK-801-induced hyperlocomotion in wild-type but not M₄ KO mice. (A) MK-801 dose-dependently increases open field locomotor activity in wild-type (WT) and M₄ KO mice. (B) In wild-type mice, IP administration of VU0467154 dose-dependently reverses MK-801-induced hyperlocomotion. (C) VU0467154 does not reverse MK-801-induced hyperlocomotion in M₄ KO mice. The time course of locomotor activity is shown on the left and total locomotor activity during the 120 min period following MK-801 administration on the right. Data are means ± SEM of 10–13 animals per group. *** p < 0.001 vs wild-type vehicle + vehicle (A); ^###p < 0.001 vs M₄ KO vehicle + vehicle (A); ^&&p < 0.01 vs. wild-type vehicle +0.3 mg/kg MK-801 (A); ⁺p < 0.05, ⁺⁺p < 0.01 vs wild-type vehicle + MK-801 (B) (ANOVA followed by Bonferroni’s test).

Figure 8

VU0467154 reverses amphetamine- (A) and MK-801-induced (B) locomotor and nonlocomotor open field activity in wild-type mice. The total number of locomotor episodes, rearings (vertical counts), and stereotypic counts and the duration of resting time during the 120 min period following amphetamine or MK-801 administration are shown. Data are means ± SEM of 7–11 (A) and 7–13 (B) mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs vehicle + amphetamine; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs vehicle + MK-801 (ANOVA followed by Bonferroni’s test).

Figure 9

Effects of MK-801 on subcortical dopamine utilization in wild-type mice are not reversed by VU0467154. Effects of single and combined treatment with MK-801 and VU0467154 on DOPAC/DA and HVA/DA ratios in the nucleus accumbens and caudate-putamen are shown. Data are means ± SEM of 9–12 animals per group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs vehicle + vehicle (ANOVA followed by Bonferroni’s test).

Figure 10

VU0467154 reverses MK-801-induced performance deficits in a touchscreen visual discrimination task. (A) Dose-dependent disruption of stable baseline performance (85–95% accuracy) of wild-type (WT) mice by MK-801. (B) Dose-dependent reversal of MK-801-induced disruption by pretreatment with VU0467154. (C). Failure of VU0467154 to reverse MK-801-induced deficits in M₄ KO mice. (D) Equal acquisition rate of pairwise discrimination in wild-type and M₄ KO mice. (E) Visual stimuli used in the pairwise discrimination task. In panels A–C, the time course of task performance is shown on the left and the performance on the test day on the right. Data are means ± SEM of 6–8 (A), 7–9 (B), 10 (C), and 9–11 (D) animals per group. ** p < 0.01, *** p < 0.001 vs wild-type vehicle + vehicle (A, B); ^##p < 0.01, ^###p < 0.001 vs wild-type vehicle + MK-801 (B); ^&p < 0.05, ^&&&p < 0.001 vs M₄ KO vehicle + vehicle (C) (ANOVA followed by Bonferroni’s test).

Figure 11

VU0467154 reverses MK-801-induced deficits in the acquisition of context-dependent fear conditioning in mice. (A) MK-801 dose-dependently disrupts the acquisition of contextual fear conditioning. (B) Pretreatment with VU0467154 reverses the MK-801-elicited deficit in contextual fear conditioning. (C) M₄ KO mice exhibit marked deficits in the acquisition of context-dependent fear conditioning. (D) VU0467154 increases the footshock threshold to elicit jumping behavior, and (E) M₄ KO mice have lower shock threshold for evoking flinching and vocalization. Data are means ± SEM of 7–10 (A), 9–10 (B), 14 (C), 6–10 (D), and 13–15 (E) animals per group. ** p < 0.01, *** p < 0.001 vs wild-type vehicle + vehicle (A, B); # p < 0.05 vs wild-type VU0467154 10 mg/kg + MK-801 0.1 mg/kg (B) (ANOVA followed by Bonferroni’s test); ^&p < 0.05 ^&&p < 0.01 ^&&&p < 0.001 vs. wild-type mice (C, E), ⁺p < 0.05 vs. wild-type vehicle (D) (t-test).

Figure 12

VU0467154 enhances the acquisition of contextual (A) and cue-dependent fear conditioning (B) in wild-type but not in M₄ KO mice. Data are means ± SEM of 10–15 wild-type and 8–13 M₄ KO mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs wild-type vehicle + vehicle (ANOVA followed by Bonferroni’s test).

Figure 13

VU0467154 does not impair rotarod performance in wild-type mice. The latency of wild-type mice to fall from a rotarod turning at 20 rpm was not affected by VU0467154. Data are means ± SEM of 10–16 wild-type mice per treatment group (ANOVA, not significant).